Composition consisting of heparin fractions having reproducible characteristics with average molecular weight equal to 5200d

ABSTRACT

Composition consisting of heparin fractions having reproducible characteristics with average molecular weight of 5,200 D obtained by depolymerization with gamma radiation and subsequent fractionation by gel permeation, having high antithrombotic properties and particularly suitable for the prophilaxis and the therapy of the alterations of the plasmatic homeostasis.

PRIOR ART

[0001] Processes to obtain the heparin and glycosaminoglycansderivatives having in general low molecular weight, for example from5,000 to 8,000 D, are known.

[0002] A process is described for example in U.S. Pat. No. 4,987,222wherein gamma radiations from Cobalt-60 are used for thedepolymerization of the heparin and other glycosaminoglycans for theachievement of derivatives having low molecular weight.

[0003] Other processes are based on chemical or enzymatic treatmentsobtained using nitrous acid, with a benzilation intervention followed byalkaline hydrolysis, digestion with isoamyl nitrite, peroxidativebreaking, the use of the specific heparinases.

[0004] The process based on the treatment with gamma radiations showsthe advantage to maintain unaltered, in the obtained fractions, theoriginary structures while in the processes based on chemical orenzymatic reactions such structures are altered whereby furthertreatments of semisynthesis and resulfation turn out to be necessary.

[0005] In any case, the problem of individuating and separatingfractions having specific activities able to intervene with aimedeffects in the different antithrombotic therapies is still open.Moreover the therapeutic use of a mixture of fractions having lowmolecular weight in order to carry on a constant activity needs acontinuative and repeatable composition of those fractions able toexpress the maximum pharmacological intervention in the absence of toxicphenomenologies typical of particular molecular weights.

SUMMARY

[0006] Now we have found a composition consisting of heparin fractionshaving high antithrombotic properties and particularly suitable for theprophylaxis and the therapy of the alterations of the plasmatichomeostasis.

[0007] Said composition is characterized by an average molecular weightequal to 5,200 D (±500) with a polydispersion index ranging from 1.04 to1.10, and characterized in that it consists of the following fractions:7,000 D (±500) 11-15% by weight 6,000 D (±500) 14-16% by weight 5,000 D(±500) 28-35% by weight 4,000 D (±500) 23-27% by weight 3,000 D (±500)13-15% by weight

[0008] Said fractions maintain unaltered the original structures of theheparin, in particular the value of the sulfation indexes constitutingthe indispensable elements for an effective therapeutic action ismaintained unaltered.

[0009] The composition according to the present invention is obtainedwith constant and reproducible characteristics from the qualitative andquantitative point of view starting from sodium heparin of animalextraction having an average molecular weight equal to 13,000-15,000 Dby treatment with gamma radiations and subsequent fractionation by gelpermeation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 represents the NMR characteristics of the compositionaccording to the present invention (GAMMAPARIN).

[0011]FIG. 2 represents the NMR characteristics of the depolymerizedmother heparin.

[0012]FIG. 3 represents the NMR characteristics of the starting heparin.

[0013]FIG. 4 represents the HPLC determination of the compositionaccording to the present invention (GAMMAPARIN).

[0014]FIG. 5 represents the HPLC determination of the depolymerizedmother heparin.

[0015]FIG. 6 represents the HPLC determination of the starting heparin.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The characteristics and the advantages of the compositionconsisting of heparin fractions obtained from depolymerization bytreatment with gamma radiation according to the present invention, willbe mostly shown during the following detailed description.

[0017] The starting material for the preparation of said compositionconsists of sodium heparin obtained from animal extraction having anaverage molecular weight equal to 13,000-15,000 D, activity equal to180-190 Ul/mg and without EDTA, heavy metals and solvents.

[0018] An aqueous solution of said heparin having a concentrationranging from 5 to 15% weight/volume is prepared.

[0019] The solution, in a neutral glass container, is piped to theirradiation cell by a system controlled by a software.

[0020] The irradiation system includes a 5 mCi radioactive source ofCobalt-60 and the heparin solution is submitted to a treatment rangingfrom 120 kGy to 150 kGy with subsequent dosages of about 25 kGy,depending on the molecular weight of the starting heparin.

[0021] The irradiated solution is treated with cut ultrafiltration 300 Dand then it is purified, concentrated, filtered and freeze-dried.

[0022] The freeze-dried product is indicated as “depolymerized motherheparin”.

[0023] The freeze-dried product is dissolved in an aqueous solution of0.3 M NaCl in such an amount to obtain a concentration ranging from 10to 15% weight/volume and the obtained solution is treated with a gelpermeation process for the fractionation.

[0024] Said process is carried out in a column containing G/50 mediumkind Sephadex resin having a granulometry ranging from 50 to 150micrometers, able to separate fractions having molecular weight rangingfrom 1,000 to 10,000 D.

[0025] For example a BP 252/15 kind pilot column may be used having thefollowing characteristics:

[0026] height: 105 cm;

[0027] resin volume: 52 l;

[0028] flux: 2 l/h of 0.3 M NaCl.

[0029] Working with the following parameters:

[0030] Ve=17 l;

[0031] Ve/Vo=1/R=1.26;

[0032] R=0.79;

[0033] K=(Ve-Vo)/(Vt-Vo)=0.09

[0034] wherein the elution volume is indicated with Ve,

[0035] the dead volume is indicated with Vo,

[0036] the retention constant is indicated with R,

[0037] the total volume of the resin bed is indicated with Vt,

[0038] 17 fractions having highly reproducible characteristics andmolecular weights ranging from values lower than 3,000 D to valueshigher than 9,000 D are usually produced.

[0039] The present invention relates to the mixture of fractions havingthe following composition: Molecular Weight % by weight 7,000 D (±500)11-15 6,000 D (±500) 14-16 5,000 D (±500) 28-35 4,000 D (±500) 23-273,000 D (±500) 13-15

[0040] This mixture represents about the 50% by weight of the total ofthe produced fractions.

[0041] Said mixture, which below will be indicated as GAMMAPARIN, showsbiological and therapeutic unexpected activities with the absence ofhaemorrhagic and allergic effects.

[0042] The chemical characteristics of GAMMAPARIN are reported in Tables1 and 2. TABLE 1 Average Molecular Weight 5,200 ± 500 PolydispersionIndex 1.04-1.10 Absorbance at 260 nm <0.200 Absorbance at 280 nm <0.1501% Solution pH 5.5-8.0 N % 1.5%-2.5% Dying Loss <5% Organic S >10% Na %9.5%-12.5% SO₃/COOH >2

[0043] TABLE 2 (with reference to the starting heparin) Ratios of thecontent in sulfated organic groups obtained by NMR analysis InitialHeparin Gammaparin (1) Desulfated Uronic 35-38% 31-34% Acids (2)GlcNSO₃6SO₃ 81-85% 83-86% (3) GlcNAc 12-15% 12-16% (4) GlcNSO₃3SO₃ 6-7%7-8%

[0044] wherein (1) is the ratio between the desulfated uronic acids withrespect to the total uronic acids, (2) is the ratio between sulfated 6glucosamine and the not sulfated one, (3) is the ratio between the Nacetylated glucosamines and the sulfated ones, (4) is the ratio betweenthe N,3 sulfated glucosamine and the not sulfated one.

[0045] These ratios confirm the validity of the depolymerizationobtained without chemical interventions of molecular adjustment.

[0046] The process based on the treatment with gamma radiationsaccording to the present invention in fact has the advantage tomaintain, in the obtained fractions, the original structures unalteredwhile in the processes based on chemical or enzymatic reactions suchstructures are often altered whereby further semisynthesis andresulfation treatments are needed.

[0047] The majority of the GAMMAPARIN components shows in the notreducing terminal of the chain a 2-O-sulfo-a-L-idopyranosuronic acid andin the reducing terminal of the chain a 2-N, 6-O-disulfo-D-glucosamine,as it is shown in the structure reported below:

[0048] with n ranging from 1 to 21, R=H or SO₃Na, R′=SO₃Na or COCH₃,R₂=H and R₃=CO₂Na or R₂=CO₂Na and R₃=H.

[0049] The process according to the present invention carries on itsintervention exclusively on the desulfated units of the structuralcomponents of the heparin molecule and it points out the possibility toobtain a high reproducibility in the formation of the derivatives havinglower molecular weights in total absence of impurities formed bycatalysts and by organic and inorganic chemical compounds extraneous tothe heparin composition.

[0050] The biological activity profiles are reported in Table No. 3TABLE No. 3 APTT antiXa antilla U/mg UaXa/mg Ualla/mg GAMMAPARIN 25-6080-100 30-50

[0051] The structural characteristics of the GAMMAPARIN, determined byNMR are reported in the FIG. 1 in comparison with the FIG. 2 of theDepolymerized Mother Heparin and in comparison with the FIG. 3 of thestarting Heparin. In the FIG. 1 (GAMMAPARIN) the signals between 84 and85 ppm characteristic of the binding site which is removed by the gammadepolymerization do not occur. The detachment of the galactosidic chainand its nitrogenous components represents a specific characteristic ofthis fraction able to work without interferences, often of pathologicalcharacter, deriving from the presence of peptidic structures havingdifferent aminoacidic composition.

[0052] The plots of the determinations in HPLC corresponding to the FIG.4 for the GAMMAPARIN, to the FIG. 5 for the Depolymerized Mother Heparinand to the FIG. 6 for the starting heparin are reported too.

PHARMACODYNAMICAL EXPERIMENTATIONS

[0053] The GAMMAPARIN antithrombotic activity has been tested accordingto “Rabbit Jugular-Vein Thrombosis Model” according to Friedman andcoll. (Am. J. Physiol. 199: 750 1960).

[0054] The intravenous administration (IV) of GAMMAPARIN from this testturned out to show a ED/50 equal to 2.15 mg/kg based on administrationsequal to 1 and 5 mg/kg showing an evident antithrombotic potential ascompared with the ED/50 of the initial heparin equal to 0.3 mg/kg. The“Bleeding Time” test has been carried out by the “Tail Transection”method according to Dejana and col. (Thromb. Haemostasis 48: 108: 1982).The bleeding times of the GAMMAPARIN in comparison with the startingheparin at the dose of 0.5 mg/kg administered 15′ before the cut areindicated in the following ranges:

[0055] Physiological Solution=155″±12.5

[0056] GAMMAPARIN=288″±15

[0057] Starting heparin=715″±43

[0058] These values point out the effect of the particular compositionof the GAMMAPARIN which shows a low bleeding index with respect to thehigh values of the heparin for the total absence of fractions having amolecular weight higher than 9,000 D.

[0059] These results confirm the structural validity of the GAMMAPARINcomponents, identifiable with the primary natural elements recognized asthe only active factors for the biological functions of the organismobtained from biological depolymerization of a highly polymerizedcomponent.

[0060] These results bear out the validity of the physical process ofthe decomposition of the heparin polymeric structure according to thepresent invention which is based on schemes of not destructiveintervention assimilable to the biological phenomena of naturalmolecular scission.

[0061] All the technical-biological data reported in the present Patentare obtained from GAMMAPARIN having sodium salt form, but they may beobtained also from the corresponding calcium, potassium, lithium,magnesium salts.

[0062] Moreover a mixture of analogous fractions may be obtainedstarting from other glycosaminoglycans such as Dermatan, Chondroitin,Heparan, Heparide and generally heparinoids.

[0063] The GAMMAPARIN may be used, as a single substance or in mixturewith other therapeutic substances, for the preparation of pharmaceuticalcompositions for subcutaneous, intramuscular, intravenous, topical andaerosol administration, in mixture with pharmaceutically acceptablesolvents or excipients.

[0064] Said pharmaceutical compositions turn out to be suitable to theprevention of the postoperative thromboembolisms, to the prophylaxis ofthe uremic patients submitted to chronical dialysis, to the preventionand the therapy of the deep venous thromboses and generally toward thealterations of the plasmatic homeostasis.

[0065] In particular, as far as the deep venous thrombosis prevention isconcerned the administration by subcutaneous injection of 100 Ul per kgof body weight every 12 hours for at least 10 days is contemplated.

[0066] For the prevention of the postoperative thromboembolisms thesubcutaneous administration of 2,500-5,000 Ul every 24 hours for atleast 7 days is contemplated.

[0067] For illustrative aim the following Examples of preparationswhich, according to the present invention, maintain constant thequali-quantitative composition of the fractions by a technology carryingout a radiation intervention as a function of the molecular weight ofthe starting heparin are reported.

EXAMPLE 1

[0068] 100 g of sodium heparin obtained from animal extraction andhaving an activity equal to 190 Ul/mg and M. W.=15,175 D (compositionpercent of the fractions in Table A) are dissolved in 1 l of bidistilledand de-aerated water. The solution is poured into a Pyrex containerwhich is closed by a glass plug after bubbling with argon.

[0069] The solution is treated with a total dose of 130 kGy atsubsequent dosages of about 25 kGy of gamma radiation from Co60.

[0070] The irradiated solution is treated with 300 D cut ultrafiltrationand purified with subsequent passages in 3% NaCl. The solutionconcentrated at 10% is freeze-dried and the freeze-dried product(Depolymerized Mother Heparin No. 1) is dissolved at 10% in 0.3 M NaClsolution and fractionated with gel permeation column on G/50 mediumSephadex (batch No. 1/A).

[0071] The Table B reports the values percent of the obtained fractions.TABLE A Batch n.1 - MW 15,175 D MW (KD ± 500) % Fractions >20 23.68 15 ÷20 23.18 15 ÷ 12 14.53 12 ÷ 10 11.74 10 ÷ 8  12.41  8 ÷ 6 7.42  6 ÷ 45.53  4 ÷ 2.5 1.47 <2.5 0.04

[0072] TABLE B Batch n.1/A - MW 6,460 D MW (D ± 500) % Fractions >9,00023.21 8,000 7.55 7,000 8.41 6,000 9.05 5,000 18.90 1,000 17.10 3,0009.31 <3,000 6.40

[0073] The preparation of the composition according to the presentinvention (GAMMAPARIN) is obtained by the mixing of the fractionsranging from 7,000 D to 3,000 D in order to obtain an average molecularweight equal to 5,200 D (±500). The mixture in an aqueous solution at aconcentration equal to 5 g/l is submitted to desalting on 1,000 Dcut-off membranes and then it is concentrated to a concentration equalto 50 g/l. The concentrated solution is freeze-dried after undergoingsterile filtration.

[0074] About 40 g of a composition with the following characteristicsare obtained:

[0075] Average M. W. 5,200 D (±500)

[0076] Molecular Weights Distribution: <3,000 D=1.5% ->7,000 D=8%

[0077] Polydispersion Index: <1.1

[0078] Sulfates/Carboxyl Ratio: >2

[0079] AntiXa Activity: >80 U antiXa/mg.

EXAMPLE 2

[0080] 100 g of sodium heparin obtained from animal extraction andhaving an activity equal to 186 Ul/mg and M. W.=14,200 Da (compositionin Table C) are dissolved in 1 l of bidistilled and de-aerated water.The solution is poured into a Pyrex container and bubbled with argon.The closure of the container by a glass plug is immediate. The solutionis treated with a total dose of 125 kGy at subsequent dosages of gammaradiation from Cobalt 60. The operations described in the Example No. 1follow obtaining the Depolymerized Mother Heparin which is fractionated(batch No. 2/A) with the percentages of the fractions of Table D. TABLEC Batch n.2 - MW 14,200 D MW (KD ± 500) % Fractions >20 20.18 15 ÷ 2018.80 15 ÷ 12 20.05 12 ÷ 10 11.70 10 ÷ 8 9.64  8 ÷ 6 10.69  6 ÷ 4 6.76 4 ÷ 2.5 2.08 <2.5 0.20

[0081] TABLE D Batch n.2/A - MW 6,944 D MW (D ± 500) % Fractions >9,00027.20 8,000 7.77 7,000 8.45 6,000 9.08 5,000 16.10 4,000 15.75 3,0008.24 <3,000 5.56

[0082] The preparation of the composition according to the presentinvention (GAMMAPARIN) is obtained by the mixing of the fractionsranging from 7,000 D to 3,000 D in order to obtain an average molecularweight equal to 5,200 D (±500). The operations described in the ExampleNo. 1 follow and a product having the same characteristics is obtained.

EXAMPLE 3

[0083] 100 g of sodium heparin obtained from animal extraction andhaving an activity equal to 185 Ul/mg and M.W.=12,910 D (composition inTable E) after the dissolution operations, are irradiated with a totaldose equal to 120 kGy according to the conditions of the Example 1. TheDepolymerized Mother Heparin, after fractionation with gel permeation(batch No. 3/A) shows the fractions indicated in Table F. TABLE E Batchn.3 - MW 12,910 D MW (KD ± 500) % Fractions >20 16.36 15 ÷ 20 21.23 15 ÷12 14.98 12 ÷ 10 12.67 10 ÷ 8 9.86  8 ÷ 6 10.10  6 ÷ 4 7.17  4 ÷ 2.53.71 <2.5 3.72

[0084] TABLE F Batch n.3/A MW 6,800 D MW (D ± 500) % Fractions >9,00026.98 8,000 6.89 7,000 7.27 6,000 9.08 5,000 19.64 4,000 12.96 3,0008.30 <3,000 7.88

[0085] The preparation of the composition according to the presentinvention (GAMMAPARIN) is obtained by the mixing of the fractionsranging from 7,000 D to 3,000 D in order to obtain an average molecularweight equal to 5,200 D (±500). The operations described in the ExampleNo. 1 follow and a product having the same characteristics is obtained.

1. Composition consisting of heparin fractions having reproduciblecharacteristics obtained by depolymerization with gamma radiation andsubsequent fractionation by gel permeation, having an average molecularweight equal to 5,200 D (±500) and consisting of 11-15% of the fractionhaving molecular weight equal to 7,000 D (±500), of 14-16% of thefraction having molecular weight equal to 6,000 D (±500), of 28-35% ofthe fraction having molecular weight equal to 5,000 D (±500), of 23-27%of the fraction having molecular weight equal to 4,000 D (±500) and of13-15% of the fraction having molecular weight equal to 3,000 D (±500).2. Composition as claimed in claim 1, having a polydispersion index ofthe molecular weights ranging from 1.04 to 1.1.
 3. Composition asclaimed in claim 1, having an absorbance at 260 nm<0.200 and anabsorbance at 280 nm<0.150.
 4. Composition as claimed in claim 1,wherein it maintains the sulfation values corresponding to the values ofthe starting heparin.
 5. Composition as claimed in claim 1, having anorganic sulphur content >10%.
 6. Composition as claimed in claim 1,wherein the galactosidic chain and its nitrogenous components present inthe starting heparin structure are absent.
 7. Composition as claimed inclaim 1, having an activity in APTT, antiXa and antilla respectively of25-60 U/mg, 80-100 UaXa/mg and 30-50 Ualla/mg.
 8. Composition as claimedin claim 1, having an antithrombotic activity, ED/50 of 2.15 mg/kg. 9.Composition as claimed in claim 1, having a bleeding index of 288±15sec. at the dose of 0.5 mg/kg.
 10. Composition as claimed in claim 1,being salified with a cation selected from the group consisting ofsodium, potassium, lithium, calcium and magnesium.
 11. Composition asclaimed in claim 1, wherein said fractions derive from Dermatan,Chondroitin, Heparan, Heparide and heparinoids.
 12. Pharmaceuticalcompositions suitable to the prophylaxis and the therapy of theplasmatic homeostasis alterations containing an effective amount of thecomposition as claimed in claim 1, either as a single substance or inassociation with other therapeutic substances.
 13. Pharmaceuticalcompositions including an effective dose of the composition as claimedin claim 1 in mixture with solvents or pharmaceutically acceptableexcipients, in a form suitable to the intravenous, intramuscular,subcutaneous, intradermic administration or in ointment, gel or sprayshape, suitable to the prophylaxis and the therapy of the plasmatichomeostasis.